Development of a validated UPLC–MS/MS method for PK/PD analysis of SYL930 and its two major metabolites in dogs

A sensitive and specific UPLC‐MS/MS method was developed and validated for the simultaneous determination of 2‐amino‐2‐(2‐(4′‐(2‐propyloxazol‐4‐yl)‐1,1′‐biphenyl]‐4‐yl)ethyl)propane‐1,3‐diol (SYL930), phosphorylated metabolite (SYL930‐P) and hydroxylated metabolite (SYL930‐M) in dog blood using SYL927 and SYL927‐P, analogues of SYL930, as the internal standards. Analytes were extracted with protein precipitation followed by chromatographic separation on a ZorbaxSB‐C₁₈ column (3.5 μm, 2.1 × 100 mm) with a gradient elution of methanol–water containing 0.1% formic acid (v /v). A triple quadrupole tandem mass spectrometer operating in the positive electrospray ionization mode was used to detect SYL930, SYL930‐P, SYL930‐M and IS transitions of 381.2 → 364.2, 461.2 → 334.2, 397.3 → 380.3, 367.1 → 350.4 and 447.5 → 320.2, respectively. The linear calibration curves for SYL930, SYL930‐P and SYL930‐M were 0.5–500, 0.2–100 and 0.5–100 ng/mL, respectively (r ² > 0.99). The intra‐day and inter‐day precisions (RSD, %) of analytes did not exceed 9.16% except for low QCs (≤16.22%), and the accuracy (RE, %) ranged from −14 to 11.4%. The mean recoveries for SYL930, SYL930‐P and SYL930‐M in dog blood were 85.13–107.94, 73.84–80.08 and 85.64–95.44%, respectively. The validated method was successfully applied to pharmacokinetic and PK/PD studies of SYL930 and its two major metabolites in dogs after an oral administration of SYL930.